The development of methods that rapidly and accurately identify interactions between structurally diverse proteins and/or peptides is key to advancing the growing field of proteomics. Identifying interactions involving structurally diverse proteins with broad biophysical properties is critical to expanding our understanding of complex cellular processes. Techniques such as immuno-precipitation, mass spectrometry, affinity purification, and protein microarrays have been used to identify interactions involving proteins and/or peptides in vitro. However, these approaches are laborious (typically requiring distinct expression and purification steps for each protein or peptide studied), low-throughput, are often limited to high-affinity interactions, and can involve complicated and/or expensive equipment. Perhaps most importantly, these methods do not provide any strong information on the likelihood of identified interactions occurring in vivo.
Popular in vivo approaches to identify and study interactions involving proteins and/or peptides include two-hybrid screening and split-protein reassembly. Common limitations of the two-hybrid screening approach include: the significant number of false positive results; the need for nuclear localization of the interacting proteins or peptides; and the need for transcription and translation of the reporter protein, which increases the overall length and complexity of the two-hybrid screening approach.
Split-protein reassembly has been used as an alternative to two-hybrid methods to identify and study protein-protein interactions within prokaryotic and eukaryotic cells using a reporter protein that is split into two fragments and fused to possible interacting peptide and/or protein partners in the protein-protein interaction of interest. In the absence of fused binding partners, the split-reporter fragments do not reassemble and reporter activity is not observed. However, if the interacting peptide and/or protein partners have sufficient affinity for one another, the resulting protein-protein interaction brings the two fragments of the split reporter protein, resulting in the reassembly of a functional reporter protein and associated reporter activity.
In general, reporter proteins typically fluoresce, catalyze a colorimetric or fluorescent reaction, or endow a host cell with resistance to an exogenous toxin. Split-reporter proteins currently used to detect protein-protein interactions in bacteria, S. cerevisiae, and mammalian cells include β-lactamase, β-galactosidase, dihydrofolate reductase, ubiquitin, and Green Fluorescent Protein (GFP). GFP is a particularly well-suited split-reporter protein for at least several reasons. GFP does not require the addition of exogenous reagents in order to generate a signal. In addition, GFP expresses, folds, and fluoresces in a large number of cell types and intracellular compartments, and is generally resistant to proteolytic degradation in vivo. Further, the formation of a fluorescent chromophore in GFP is an irreversible reaction, enhancing the ability of reassembled GFP reporter protein to detect weak protein-protein interactions with dissociation constants (Kd) as high as 1 mM.
However the use of GFP as a split-reporter protein is not without some limitations. The fragments of the split GFP reporter protein may be susceptible to instability and aggregation within the cell during use. Some existing split-GFP reporter proteins, such as split-sg100 GFP, incorporate enhanced-stability GFP variants. Even using these enhanced-stability GFP variants, interaction-dependent reassembly screens using split-sg100 GFP fusions may be performed well below physiological temperature (typically 20° C.-30° C.) to further enhance fragment stability to acceptable levels. However, the results of interaction-dependent reassembly screens conducted at reduced temperatures may not be applicable at physiological temperatures due to the sensitivity of protein-protein interactions to changes in ambient temperature. In addition, existing stabilized split-GFP reporter proteins, such as split-sg100 GFP, may requires 24-72 hours in order to generate visible levels of cellular GFP fluorescence.
A need exists for a split-reporter protein in which the protein fragments are stabilized and resistant to aggregation at physiological temperatures. In addition, a need exists for a robust split-reporter protein capable of interaction-dependent reassembly under a variety of conditions and further capable of generating a reporting signal in a relatively short time.